13 research outputs found
Ariel - Volume 2 Number 7
Editors
Richard J. Bonanno
Robin A. Edwards
Associate Editors
Steven Ager
Stephen Flynn
Shep Dickman
Tom Williams
Lay-out Editor
Eugenia Miller
Contributing Editors
Michael J. Blecker
W. Cherry Light
James J. Nocon
Lynne Porter
Editors Emeritus
Delvyn C. Case, Jr.
Paul M. Fernhof
Ariel - Volume 2 Number 6
Editors
Richard J. Bonanno
Robin A. Edwards
Associate Editors
Steven Ager
Stephen Flynn
Shep Dickman
Tom Williams
Lay-out Editor
Eugenia Miller
Contributing Editors
Michael J. Blecker
W. Cherry Light
James J. Nocon
Lynne Porter
Editors Emeritus
Delvyn C. Case, Jr.
Paul M. Fernhof
Ariel - Volume 4 Number 2
Editors
David A. Jacoby
Eugenia Miller
Tom Williams
Associate Editors
Paul Bialas
Terry Burt
Michael Leo
Gail Tenikat
Editor Emeritus and Business Manager
Richard J. Bonnano
Movie Editor
Robert Breckenridge
Staff
Richard Blutstein
Mary F. Buechler
Steve Glinks
Len Grasman
Alice M. Johnson
J. D. Kanofsky
Tom Lehman
Dave Mayer
Bernie Odd
Cooperativity in the Binding of Agonists to Reconstituted Tetramers of the M2 Muscarinic Receptor
Efficacy as an Intrinsic Property of the M<sub>2</sub> Muscarinic Receptor in Its Tetrameric State
Muscarinic and other G protein-coupled
receptors exhibit an agonist-specific
heterogeneity that tracks efficacy and commonly is attributed to an
effect of the G protein on an otherwise homogeneous population of
sites. To examine this notion, M<sub>2</sub> muscarinic receptors
were purified from <i>Sf</i>9 cells as monomers devoid of
G protein and reconstituted as tetramers in phospholipid vesicles.
In assays with <i>N</i>-[<sup>3</sup>H]Âmethylscopolamine,
seven agonists revealed a dispersion of affinities indicative of two
or more classes of sites. Unlabeled <i>N</i>-methylscopolamine
and the antagonist quinuclidinylbenzilate recognized one class of
sites; atropine recognized two classes with a preference that was
the opposite of that of agonists, as indicated by the effects of <i>N</i>-ethylmaleimide. The data were inconsistent with an explicit
model of constitutive asymmetry within a tetramer, and the fit improved
markedly upon the introduction of cooperative interactions (<i>P</i> < 0.00001). Purified monomers appeared to be homogeneous
or nearly so to all ligands except the partial agonists pilocarpine
and McN-A-343, where heterogeneity emerged from intramolecular cooperativity
between the orthosteric site and an allosteric site. The breadth of
each dispersion was quantified empirically as the area between the
fitted curve for two classes of sites and the theoretical curve for
a single class of lower affinity, which approximates the expected
effect of GTP if a G protein were present. The areas measured for
10 ligands at reconstituted tetramers correlated with similar measures
of heterogeneity and with intrinsic activities reported previously
for binding and response in natural membranes (<i>P</i> ≤
0.00002). The data suggest that the GTP-sensitive heterogeneity typically
revealed by agonists at M<sub>2</sub> receptors is intrinsic to the
receptor in its tetrameric state. It exists independently of the G
protein, and it appears to arise at least in part from cooperativity
between linked orthosteric sites